Method for preventing respiratory mycoplasmosis of livestocks

ABSTRACT

Respiratory mycoplasmosis of livestocks such as fowl chronic respiratory disease, swine enzootic pneumonia and contagious pleuropneumonia of goats are effectively immunised by spraying or injecting into tracheas of the livestocks killed Mycoplasma vaccine which is a suspension of killed Mycoplasma cells, with suspension stabilizer such as gelatin, serum of the livestocks, saccharides, amino acids and Tween 80.

United States Patent [191 Yoshioka et al.

[4 1 Nov. 4, 1975 METHOD FOR PREVENTING RESPIRATORY MYCOPLASMOSIS OF LIVESTOCKS [75] Inventors: Morimasa Yoshioka; Eizo Hayatsu,

both of Tokyo, Japan [73] Assignee: The Kitasato Institute, Tokyo, Japan [22] Filed: Sept. 17, 1973 [21] .Appl. No.1 397,909

[30] Foreign Application Priority Data OTHER PUBLICATIONS Adler et al., V.B. No. 3893 (1971), Abstract of immunization Against Mycoplasma Gallisepticum,

Avian, Dis. 14: 763-769 (1970).

Papageorgiou, V.B. No. 5668 (1971), Abstract of Immunization Against Avian Mycoplasmosis, I. Attentuation of a Strain of Mycoplasma Gallisepticum, 11. Use of the Live Attenuated Vaccine, Bull. Acad. VBT, Fr. 43: 363-375 (1970).

Primary Examiner-Shep K. Rose Attorney, Agent, or FirmArmstrong, Nikaido & Wegner [57] ABSTRACT Respiratory mycoplasmosis of livestocks such as fowl chronic respiratory disease, swine enzootic pneumonia and contagious pleuropneumonia of goats are effectively immunised by spraying or injecting into tracheas of the livestocks killed Mycoplasma vaccine which is a suspension of killed Mycoplasma cells, with suspension stabilizer such as gelatin, serum of the livestocks, saccharides, amino acids and Tween 80.

18 Claims, N0 Drawings METHODFQR PREVENTING RESPIRATORY MYCOPLASMOSIS F LIVESTOCKS' This invention relates to killed vaccines for immunizing livestocks from respiratory infectious disases caused by Mycoplasma, and to a process for preparing thesame.

Heretofore, respiratory mycoplasmosis of livestocks have been prevailed in all countries of the world. There are such diseases as chronic respiratory diseases of [30111115108 caused by Mycbplasmq gallisepticum, sacculitis of poultriescaused by Mycoplasma synoviae, enzootic'pneurnonia of swine caused by Mycoplasma suipn eumonzja e, .contagious pleuropneumonia of goats caused by .MycbpIz 1s'ma mycoides var. capri, etcQThese enzootie respiratory Mycoplasma diseases of livestocks are highly contagious, and itis well known that as the resultof infection with one or more of said Mycoplasmadiseasjes, the ratios of body weight increase,.

egg-production and fertilization are lowered, and the numbers of was techickens areincreased in poultry. feed efficiency and body weightincrease ratio are decreased in hogs, grow weakand die of diarrhea caused by the said disease in goats. Therefore, the poultry industry. anad stock-raisers are suffering from a great economical damage through the said mycoplasmosis. Accordingly, the establishment of countermeasures has been desired for preventing the said enzootic respiratory diseases, causedby Mycoplasma, i 7

With the aimto cure the enzootic respiratoryMycoplasma diseases of vlivestocks, several antibiotics are tried and used, but it is extremely difficult to eradicate the aforesaid diseases by the use of antibiotics. For immunization for the said diseases, living vaccines using attenuated strains have been proposed [U.S. Pat. No. 3,534,136 (1970)]. As the back fo the virulence and the vertical transmission of the vaccine organisms are the problemslto be throughly clarified inliving vaccines, the development of effective and safe inactivated vaccines is desired. As, to inactivated vaccines, there have been reports concerning various adjuvant vaccines which is administered by intramuscular injection [McMartin, D. A., and. Shifrine,.M. :Am. J. of Veter. Res, Vol. 21, page 482 (1960); Adler, H. E.,- Fabricant, ,l., Yamamoto, R. and Berg. J. Am. J. of Veter. Res, Vol 19, page 440 (1958)]. However, all these adjuvant vaccines cannot sufficiently prevent the said diseases, andhence have notbeen put into wide use.

An object of the present invention is to provide an immunization procedure for Mycoplasma diseases in livestocks.

Another object of the=present invention is to provide a killed vaccine acceptable for the immunization pro-.

traches of the livestocks and a killed Mycoplasrna vaccinesuspension therefor.

The killed Mycoplasmav vaccine used thejpiresent.

invention is prepared by cultivating Mycoplasma or-,

fact that the Beef heart influsion 0s Peptone y v 10.0 g Sodium'chloride 5.0 g

' Water to make 1000 ml The liquidrnedium is addi tionally incorporated with a suitable carbori source such as glucose in an amount of 0.1 to 1.0% wt/vol. In case of the cultivation of Mycoplasma gallisepticum poultry serum is further added in an amount of 5 to 15% by volume. In case of the cultivation of Mycoplasma synoviae, the medium is further added with 5 to 15% of poultry serum, 0.001 to 0.1% wt/vol. of diphosphopyridine nucleotide, 0.001 to 0.1% of L-cysteine hydrochloride and 0.01 to 0.1% wt/vol. of Eagle vitamins X concentrate)*.

[*A trade mark for multi-vitamine concentrate preparation used for culture medium. 'Manufactured' by and sold from Difco' Laboratories Inc, Detroit;

Michigan, U.S.A. One liter of the concentrate contains:

' cl-Biotin 0.24 mg Folic acid 0.44 mg Niacinamide I I 0.12 mg I Calcium pantothenate 0.24 mg Pyrid'oxal hydrochloride (U.S.P.) 0.20 mg Thiamine hydrochloride 0.34 mg Rihoflavine 0.04 mg Choline chloride 0.l4 mg For the cultivation of ll lyc 'oplasma suipneuhwniae, the medium is further added with 10 to 20%. wt/vol. of

swineserum; and for the cultivation of Mycoplqsn za mycoides var. capri, the medium is further added with 5 phosphatebuffer solution or a mixture of M/100 of phosphate buffered physiological saline solution. The extraction can .becarried out, for example, by stirring a mixture of egg yolk and deionized water (1 l by volume) at 56C for 2 hours, and then cooled tea room temperature. The mixture is then centrifuged at 10,000

rpm. and the resulting supernatant fluid is filtered under a sterile condition to obtain an egg yolk component.

Each of the aforesaid Mycoplasma strains is inoculated into the thus prepared Mycoplasma cell propagation liquid culture medium and cultured aerobically at 3538C for l to 7 days to obtain Mycoplasma cells in a high yield eg 10 g to 5.0 g of the ,wet cells per 1 liter of the cultured liquid medium.

In order to harvest the Mycoplasma cells, the cultured liquid medium is centrifuged by the use of a centrifugal separator at 8000 rpm. or more for 20 minutes or more or by the use of a continuous centrifugal separator, for example, at 16000 rpm (about 25000 G).

Alternatively, the cultured liquid medium is added with an aluminum hydroxide gel or an aluminum phosphate gel as an adsorbent to separate or precipitate the Mycoplasma cells. The amount of the adsorbent used is in ranging from 10 to 400 ml, preferably from 30 to 100 ml, per 1 liter of the cultured liquid medium.

The cells thus harvested are then added with a physiologically acceptable aqueous medium having a pH of 6.0 to 7.8, preferably a pH of 6.5 to 7.4, such as physiological saline solution, M/ l of a phosphate buffer solution or a mixture of M/ l 00 phosphate buffered physi- .ological saline solution to obtain a cell suspension of NaHPO 958 mg NaCl 8.5 g Water to make 1,000 cc. pH 7.2

In the above manner, the obtained cell suspension is adjusted to such a concentration that the number of cells per ml becomes more than 2.0 X 10 The resulting cell suspension is heated at 50 to 56C for 5 to 30 minutes, or is added with a killing agent in an amount to be added as indicated for example, such as formalin (0.01 to 0.5% wt/vol), sodium ethylmercury salicylate (0.01 to 0.02% wt/vol) or B-propiolactone (0.01 to 0.2% wt/vol) to kill the cells.

Alternatively, the precipitate is suspended in a small amount of physiological saline solution, and the resulting suspension is subjected to such inactivation treatment as to completely destroy the Mycoplasma cells by means of a physical operation such as ultrasonic disintegration at kilocycles per second for 0.5-1.0 minute or French pressing, and then diluted with the physiological saline solution to attain the above-mentioned cell concentration, thereby obtaining a killed cell suspension.

'The thus prepared inactivated Mycoplasma vaccine is a suspension of killed Mycoplasma cells or its fractions in a physiological saline solution of pH 6.0 to 7.8, the number of cells or fractions corresponding to at least 2.0 X 10 per ml of thesuspension of living cell.

An excellent immunization effect can be obtained even whenthe killed cell suspension is used also, but to enhence its effect, the suspension may preferably contain a suspension stabilizer.

As to the suspension stabilizer, one or mixtures of the below-mentioned physiologically acceptable substances are usable, in the amount as indicated.

Saccharides: Glucose (0.3-1 .0%

(0.5-1.0%), lactose (0.1-1.0%), or (0.5-3.0%).

Amino acids: Glutamic acid (0.1-0.5%), or glycine Serums:

In the case of Mycoplasma gellisepticum or Mycoplasma synoviae: Poultry serum (0.1-1.0%), or poultry serum albumin (0.1-0.5%). In the case of Mycoplasma suipneumoniae:

sucrose dextran Swine serum (0.1-1.0%), or swine serum albumin (0.1-0.5%).

4 In the case of Mycoplasma mycoides var. capri:

Goat serum (0.1-1.0%), or goat serum albumin Others:

Tween (ODS-0.5%), gelatin(0.0l0.05%), or disodium ethylenediamine-tetraacetate (0.03-0.3%). (*Tween 80: A trademark for sorbitan monooleate of polyoxyethylene condensate. Manufactured by and sold from Atlas Chemical Industries Inc., Chemicals Division, Wilmington, Del., U.S.A.

When the suspension stabilizer is added to the inactivated cell suspension, the cell suspension is homogenized by the use of any physical methods; for example, an ultrasonic treatment at 10 kilocycle'per second for 30 seconds to 1 minute or shaking with glass beads, and then the suspension stabilizer as mentioned above is added in a suitable amount into the cell suspension to obtain the suspension of killed Mycoplasma vaccine.

The prevention of respiratory diseases caused by infection with Mycoplasma is effected through immunization of respiratory organs of livestocks by spraying or injecting the vaccine suspension into tracheas.

It should be noted the fact that the suspension of killed Mycoplasma vaccine of the present invention cannot provide any distinct preventive effect when the vaccine is intramusclularly injected either alone or in admixture with various adjuvants such as, for example, Bacto-Adjuvant, Complete Freund magnetic iron oxide, a mixture of sodium alginate and calcium chloride, a mixture of inactivated Hemophilus gallinarum and aluminum hydroxide gel or an aluminum hydroxide gel.

A trademark for an adjuvant of the following composition:

Arlacel A "(Mannite monooleate) 1.5 ml

Bayol F (Paraffin oil) 8.5 ml

Mycobacterium butyricum (killed and dried). Manufactured by and'sold from Difco Laboratories, Inc., Detroit, Michigan, 48201, USA.

1.A trademark for a nonionic emulsifier for use in pharmaceuticals. It is a fatty acid partial ester of polyol anhydride. Manufactured by and sold from Atlas Chemical Industries, Inc. Wilmington, Del., 19899, U;S.A. v

2. A trademark for a technical-grade white mineral oil that is used where U.S .P. or N.F. quality is not required. Manufactured by and sold from Humble oil & Refining Co., Houston, Texas, 77001, (U.S.A.)]

The suspension of inactivated Mycoplasma'vaccine of the present invention, however, can give an excellent preventive effect when it is independently inoculated into the traches by spraying or tracheal injection. Thus the immunization procedure carried out by spraying or injecting the present vaccine into traches is a safe immunization procedure bringing no such side-effects as induration of injected sites which have frequently been observed in the case of intramuscular injection of the prior art inactivated vaccines.

The amount'of the present suspension of killed Mycoplasma vaccine to be administered by spraying or by injecting into tracheas may vary depending upon the degree'of the infection of livestocks. 0.1 milliliters or more amounts of the suspension of killed Mycoplasma gallisepticum or Mycoplasma synoviae vaccine or mix ture thereof can be used for preventing air sacculitis of poultries: 0.5 ml or more amounts of the suspension of killed Mycoplasma suipneumoniae vaccine can be used for preventing enzootic pneumonia of pigs; 0.5 ml or more amounts of the suspension of killed Mycoplasma mycoides var. capri can be used for preventing contagious pleuropneumonia of goats. Usually, the vaccines may be administered by spraying or injecting into tracheas by the use of nebulizer or any suitable spraying apparatus, 2 or 3 or even more times at intevals of days.

Suspension of killed Mycoplasma vaccines of the present invention show significant effects on immunizing the infection with virulent Mycoplasma strain, even if the livestocks being immunized with the vaccine are further infected with other pathogenic microorganisms.

The present invention is illustrated in detail below with reference to examples, but the scope of the invention is not limited to the examples.

EXAMPLE 1 One liter of the basic Mycoplasma cultivation liquid medium was added with 0.5% wt/vol of glucose and 10% by volume of heated chicken serum to prepare a cell propagation liquid culture medium. Subsequently, Mycoplasma gellisepticum was inoculated in the medium, and cultured under aerobic conditions at 35 to 38C for 2 days. After the cultivation the cultured liquor was centrifuged at 13,000 rpm. for 30 minutes to.

obtain a precipitate of Mycoplasma gallisepticum cells.

To this precipitate was added an aqueous phosphate EXAMPLE 2 Example 1 was repeated, except that the heated chicken serum among the components of the Mycoplasma gallisepticum cell propagation medium was replaced by 6% by volume of egg yolk component. extracted with deionized water. There was obtained a suspension of killed vaccine of Mycoplasma gallisepticum.

EXAMPLE 3 Example 1 was repeated, except that the centrifugal separation of Mycoplasma gallisepticum cells was replaced by adding 50 ml of aluminum hydroxide gel to 1000 ml of the cultured medium. There was obtained a suspension of killed vaccine of Mycoplasma gallisepticum.

EXAMPLE 4 6 of L-cysteine hydrochloride and 0.025% Eagle-Vitamins X concentrate) were added. There was obtained a suspension of killed vaccine of Mycoplasma synoviae.

EXAMPLE 5 Example 1 was repeated, except that Mycoplasma gallisepticum and the heated chicken serum were respectively replaced by M ycoplasma suipneumoniae and 2.0% by volume of heated swineserum. Cultivation was carried out for 4 days. There was obtained a suspension of killed vaccine of Mycoplasma suipneumoniae.

EXAMPLE 6 Example 5, was repeated, except that the heated swine serum was replaced by 6% by volume of egg yolk component used in Example 2. There was obtained a suspension of killed vaccine of Mycoplasma suipneumania. I

EXAMPLE 7 EXAMPLE 8 Example 7 was repeated, except that the heated goat serum was replaced by 6% by volume of egg yolk component used in Example 2. There was obtained a suspension of killed vaccine of Mycoplasma mycoides var.

caprl.

The effect of the present method for immunization of mycoplasinosis is shown in the following Tests (Tests 1-4) in which results from spraying the present vaccine into tracheas and intramuscular injection of the present vaccine with. anadjuvant are compared.

Test 1 Ten specific pathogen-free (SPF) chickens of 10 days old in one test-group were immunized by spraying into respiratory tract with the suspension of killed vaccine of Mycoplasma gallisepticum obtained in Example 1.

Another 10 SPF-chickens of 10 days old in the second group were immunized by intramuscular injection with the same suspension of killed vaccine used in the first group.

Further, 10 SPF-chickens of 10 days old in other groups were immunized by intramuscular injection with each of a various adjuvants with the same suspension of killed vaccine used in the first group. The immunizations were carried out 3 times at intervals of 10 days. Immunization dose at one time was 0.1 ml. Ten days thereafter, all the test chickens in three groups were challenged by spraying with varulent Mycoplasma gallisepticum strain having 3.2 X 10 or 1.6 X 10 living cells. Two weeks after the challenge, the test chickens were sacrified and examined to evaluate the effects of the vaccines according chiefly to the macroscopic lesions of air sacs. The results are shown in Table 1.

Table l Challenge dose Vaccine Adjuvanl Route (Number of Lesion score living cells) of air sacs 1n the 3.2 X 10 Table l-continued Challenge dose Vaccine Adjuvant Route (Number of Lesion score living cells) of air sacs* muscles 1.6 X 10 1.80 Bacto-Adjuvant. 3.2 X 10 2.75 Suspension of Complete Freund 1.6 X 2.10 killed Magnetic iron oxide 3.2 X 10 2.80 vaccine of 1.6 X 10 2.00 Mycoplasmu Sodium alginate 3.2-x 10 2.70 gallisepticimi Calcium chloride 1.6 X 10 1.90 obtained in lnactivated Hemophilus 3.2 X 10 3.00 Example 1. gallinarum Aluminum 1.6 X 10 2.05

hydroxide gel Aluminum hydroxide gel 3.2 X 10 2.70 1.6 X 10* 1.85 lntra- 3.2 X 10 1.75 tracheas 1.6 X 10 1.40 3.2 X 10 3.15 1.6 X 10 2.00

0.60 Control (Normal) (Average value of scores showing the degree of lesion of air saces regarding the respective test groups consisting of 10 chickens. The score of each chickens is obtained as an average value of the score of degree of the disease of four air sacs. The degrees of lesion is evaluated according to the state of air sacs as shown below:

: Nonnal Thickness of air sacs Focal lesions I Formation of even one caseous lump.

: Formation of many or overall caseous lumps) As is clear from Table 1, it has been found that the above-mentioned suspension of killed vaccine cannot provide any distinct preventive effect when intramuscularly immunized either alone or in admixture with various adjuvants, but can give the preventive effect by spraying with virulent Mycoplasma suipneumoniae having 5.5 X 10 living cells. After 6 weeks, the test pigs were dissected to evaluated the effects of the vaccines according to pathological observation on the lungs. The results obtained are shown as in Table 3.

wliistnzit is directly inoculated into the trachea. Table 3 Test 1 was repeated, except that the suspension of lnfec killed vaccine of Mycoplasma gallisepticum and the virvaccine Adjuvam Rome gm f w I on ulent Mycoplasma galli'septicum were respectively remg cells) rate* placed by the suspension of killed vaccine of Myco- 1mm X 9 8/10 plasma synom'ae obtained in Example 4 and virulent B t musclar ac Mycoplasma synovzae having 5.7 X 10 living cells. The 40 Adj-yam. H 7/10 results obtained are shown in Table 2. Complete Table 2 Challenge dose Lesion score Vaccine Adjuvant Route (Number of of air sacs* living cells) 1n the 5.7 X 10 2.20

, muscles Suspension Bacto-Adjuvam, 2.25 of killed Complete Freund vaccine of Magnetic iron oxide 2.30 Mycuplasma Sodium alginate 2.25 synoi'iae Calcium chloride obtained in inactivated Hemopliilus 2.20 Example 4 gallinarum Aluminum hydroxide gel Aluminum hydroxide gel 2.20 Intra 1.55

trachea Control 2.35

(Same as in Table 1) Test 3 r Suspension Freund of killed Magnetic Test 1 was repeated, except that the suspension of vaccine of iron oxide N 8H0 killed vaccine of Mycoplasma gallzseptzcum, the viru- Mtcoplasn a Sodium lent Mycoplasma gallisepticum and SPF-chickens were f 9/10 obtained in Calcium respectively replaced by the suspension of killed vac- Emmph 5 cmmide cine of Mycoplasma suipneumoniae obtained in Exam- 2 g"? emu ll 8 pie 5, virulent Mycoplasma suipneumoniue having 5.5 X guumlfmm 8/] 0 10 living cells, and SPF-pigs of 3 to 4 weeks old. The Aluminum immunization dose of one time was 0.5 ml. Ten days 333 2232 after the final immunization, the pigs were challenged hydroxide gel 9 10 Table 3-continued Challenge dose (Numlnfec- Vaccine Adjuvant Route ber of livtion ing cells) rate* lntra- 4/ 1O trachea Control 9 (The denominator shows the number of test pigs. and the numerator shows the number of lung disease-positive pigs.)

Test 4 Test 3 was repeated, except that the suspension of killed vaccine of Mycoplasma suipneumoniae, the virulent Mycoplasma suipneumoniae and the SPF-pigs were respectively replaced by the suspension of killed vaccine of Mycoplasma mycaides var. capri obtained in Example 7, virulent Mycoplasma mycoides var. capri having 7.0 X 10 living cells and SPF-goats of 3 week old. The results obtained are shown in Table 4.

(The denominator shows the number of test goats. and the numerator shows the number of lung disease-positive goats.)

In order to know the preventive effect of the suspension of killed vaccine of Mycoplasma on respiratory diseases of livestocks caused by contagion with virulent Mycoplasma-infected livestocks, the following tests (Tests -9) were conducted.

Test 5 SPF-Chickens of days old were immunized 2 or 3 times with an intervals of 10 days with more than'0.l ml. of the suspension of killed vaccine of Mycoplasma gallisepticum obtained in Example 1 by intratracheal injection or spraying. Ten days after the final immunization, a total of 40 chickens consisting of chickens of immunized group and 20 chickens of non-immunized group (control group) were allowed to live together with 10 chickens which had been infected by spraying with virulent Mycoplasma gallisepn c'um having 2.3 X 10 living cells. After 4 weeks, the preventive effects of the inactivated vaccine on chickens respiratory Mycoplasma diseases derived from the said strain were evaluated according to the lesion grade of air sacs and to the results of isolation of Mycoplasma gallisep- 10 ticum from the tracheas, lungs and air sacs. The results obtained were as set forth in Table 5.

Table 5 Mode of Air Isolation of administration sac M yc'oplasma Total lnfecofsuspension infecgalliseplicum (-2) evalution of killed ted Tra- Air ation rate vaccine (*-1) chea Lung sac (*-3) (7:)

lntra tracheal 0/20 0/20 0/20 0/20 0/20 0 injection Spraying 0/20 0/20 0/20 0/20 0/20 0 Control 5/20 7/20 5/20 5/20 7/20 35 (*-l) The denominator shows thenumber of test chickens. and the numerator shows the number of chickens suffering from air sac disease.

(*-2) The denominator shows the number of test chickens. and the numerator shows the number of chickens. from the tracheas. lungs and air sacs of which had been isolated M \'cop!a.rmu gullixepricum.

(-3) The denominator shows the number of test chickens. and the numerator shows the number of chickens. which were suffering from air sac disease and from the tracheas. lungs and air sacs of which had been isolated Mymplm'mu gull/n lirum. i.e. the number of infected chickens.)

Test 6-1 Test 5 was repeated, except that the suspension of killed vaccine of Mycoplasma gallisepticum and the virulent Mvcoplasma gallisepticum were respectively replaced by the suspension of killed vaccine of Mycoplasma synoviae obtained in Example 4 and viluent Mycoplasma synoviae having 5.0 X 10 living cells. Contagion of the immunized group and the non-immunized group with the infected group was made 12 days after the final immunization. The results obtained are shown in Table 6-1.

Table 6-1 Mode of Air administration sac Isolation of Total Contaof suspension infec- M \'cz')pla.wna evalugion of killed ted synoi'iae (*-2) ation ratio vaccine (*-1 Trachea Air sac (*-3) (71 lntratracheal 0/ 20 0/ 20 1/20 1/20 5 injection Spraying 0/20 O/20 l/20 1/20 5 Control 3/20 5/20 11/20 8/20 40 (*-l. *-2. *-3: See the legends in Table 5) Test 6-2 Test 6-1 was repeated, except that during 7 days after the final immunization, the immunized group was 'infected with Infectious bronchitis virus and Newcastle disease virus administered by drinking water containing said viruses and then 5 days thereafter, the infected groups were allowed to live together with 10 chickens which had been infected with virulent Mycoplasma synoviae as in Test 6-1. The results obtained are shown in Table 6-2.

Table 6-2 Mode of Air administration sac lsolation of Total lnfecof suspension infec- Mycoplasma evalution of killed ted synm'iae (*-2) ation rate vaccine ("-1 Trachea Air sac (-3) (71 lntra- 0/20 0/20 2/ 20 2/20 10 tracheal injection Spraying 0/20 0/20 1/20 1/20 5 Control 6/20 5/20 9/20 9/20 45 (*-l. *2. *-3: See the legends in Table 5) Test 7-1 1 1 Test was repeated, except thatthe suspension of killed vaccine of Mycoplasma gallisepticum was replaced by a mixture of the suspension of killed vaccine of Mycoplasma gallisepticum obtained in Example 1 l2 neumaniae and the virulent Mycoplasma suipneumoniae, were respectively replaced by SPF-goats of 3 weeks old, the suspension of killed vaccine of Mycoplasma mycoides var. capri obtained in Example 7, and

and the suspension of killed vaccine of Mycoplasma 5 virulent Mycoplasma mycoides var. capri having 5.0 X synoviae obtained in Example 4. Contagion of the imliving cells. The results obtained are shown in Table munized group and non-immunized group with the in- 9. fected group was made 12 days after the final immuni- Table 9 zation. The results obtained are shown in Table 7-1.

Mode of Infection Table 7 1 Vaccine immunization rate* Mode of Air Isolation of Suspension of administration sac Mycoplasma Total lnfeckilled of suspension infecgalliseplicunl (*-2) evalution vaccine f of killed ted Tra- Air ation rate y l p y 3/10 vaccine (-1) chea Lung sac (*3) (7:) l5 "U flfi var. capri Intratriichotil 0/20 0/20 0/20 0/20 0/20 0 Comm] mm) injection Spraying 0/20 0/20 (V (V20 W20 0 *The denominator shows the number of test goats. and the numerator shows th Contro 5/20 4/20 40 20 number of lung lesion-positive goats. (-l. *-2. -3: See the legends in Table 5) Test 10 T 7-2 I In order to know the effect of a suspension stabilizer T 7-1 was repeated except h i l M to be incorporated with an killed Mycoplasma vaccine, plasma galliseplicum was replaced by virulent Mycofollowmg Comparatlve tests wem conductfad- The plasma s \'n0t'iae having 5.0 x 10 living cells. The rekilled My p a gallwepflwrrl cell suspenslpn Sults b i d are h i T bl 7.2 tamed in Example 1 and added with the suspension sta- Table 7 2 billzer, and the same killed Mycoplasma gallisepticum cell suspension without the addition of the suspension cli pp l l f T I I f stabilizer were compared. a ministration nso atlon 0 ma n ecof suspension Air sac Mycoplasma elevtion speplfic p g (.SPF) ohlckensof 10 days Old of killed infected synm'iae -Z) ation rate were immunized by spraying 3 times at intervals of 10 acme Tmche days with each of the killed cell suspension and the suslntrapension stabilizer incorporated killed vaccine (immunio/ZO 1/30 5 zation dose of one time: at least 0.1 ml.). Ten days sawing Q/ZO 0 thereafter, the chickens were challenged by spraying Control 2/20 4/20 7/20 7/20 35 with virulent Mycoplasma gallisepticum having about a see legends in Table 5 7.4 X 10 living cells. Two weeks after the challenge, the test chickens were examlned to evaluate the effects Test 8 y 40 of the vaccines chiefly by the findings of air sacs and by Test 5 was re eated exce t that the SPF chickens the results of isolation of the Mycoplasma strain from p p tracheas, lungs and air sacs. The results are shown in the suspension of killed vacclne of Mycoplasma gal- Table 10 Iiseplicum and the virulent Mycoplasma gallisepticum were respectively replaced by SPF-pigs of 3 weeks old, Table 10 the suspension of killedvaccine of [Mycoplasma suip- Lesion Score neumoniae obtained in Example 5 and virulent Mycoof chicken plasma suipneumoniae having 5.0 X 10 living cells. lm- Sac gg g l gf fzffffg munization dose of the vaccine of one-time was 0.5 ml. vaccine (an Tmhea Lung sac Six weeks after the contagion, the hogs were dissected v i lnactlvated'vacclne to investigate, accordlng to pathological observatlon on incorporated wilhou, 0.70 2H0 I no the lungs. The results obtained are shown in Table 8. suspension stabilizer Table 8 lnactivated vaccine incorporated with 0.50. 1/10 0/l0 OllO M P lnfecmm suspension stabilizer Vaccine immunization rate* suspension f Control 2.l0 10/10 10/10 8/10 2 l0 killed vaccine p y Ll) Same as in Table l. P (*-2) Number of chickens from which Mycoplasma gullisepticum was isolated. That smp'lem'mmae 1 l 0 is. the dominutor is the numberof test chickness, and the numerator is the number i P 0 of chickens from which Mymplmmla gallixeplirum was isolated. in ection Control 8/10 What is claimed is: no denominator shows the number of test pigs. nntl the numerator shows the l. A suspension of killed Mycoplasma vaccine for im- "umhcmflu'lglesw" We munizing respiratory mycoplasmosis of livestock and consisting essentially of formalin killed ultrasonically Test 9 Test 8 was repeated, except that the SPF-pigs, the suspension of killed vaccine of Mycoplasma suiphomogenized Mycoplasma cell, a physiologically acceptable aqueous medium of pH 6.0 to 7.8, and a suspension stabilizer.

2. A suspension of killed Mycoplasma vaccine according to claim 1 wherein said formalin killed and ultrasonically homogenized Mycoplasma vaccine is derived from a formalin-added cell suspension having a formalin concentration of 0.01 to 0.5%.

3. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the aqueous medium is phosphate buffered saline solution.

4. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the suspension stabilizer is a saccharide.

5. A suspension of killed Mycoplasma vaccine according to claim 4, wherein the saccharide is selected from the group consisting of glucose, sucrose, lactose and dextran.

6. A suspensison of killed Mycoplasma vaccine according to claim 5, wherein the saccharide is selected from the group consisting of lactose or dextran.

7. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the suspension stabilizer is an amino acid.

8. A suspension of killed Mycoplasma vaccine according to claim 7, wherein the amino acid is selected from the group consisting of glutamic acid and glycine.

9. A suspension of killed Mycoplasma vaccine according to claim 8, wherein the amino acid is glutamic acid.

10. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the suspension stabilizer is a serum.

11. A suspension of killed Mycoplasma vaccine according to claim 10, wherein the serum is selected from, the group consisting of chicken serum and 14 chicken serum albumin when the Mycoplasma is Mycoplasma gallisepticum.

12. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the suspension stabilizer is selected from the group consisting of sorbitan monooleate of polyoxyethylene, gelatin and disodium ethylenediamine tetraacetate.

13. A suspension of killed Mycoplasma vaccine according to claim 12, wherein the suspension stabilizer is selected from the group consisting of sorbitan monooleate of polyoxyethylene and gelatin.

14. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the number of the killed Mycoplasma cells is at least 2.0 X 10 per ml of the vaccine.

15. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the killed Mycoplasma cell is Mycoplasrria g allisepticum cell and the livestock is poultry.

16. A method of immunization for respiratory infections Mycoplasma diseases of livestocks comprising administering a formalin killed and ultrasonically homogenized Mycoplasma vaccine to the livestocks by spraying or injecting the killed vaccine into trachea.

17. A method according to claim 16 wherein said formalin killed and ultrasonically homogenized Mycoplasma vaccine is derived from a formalin-added cell suspension havinga formalin concentration of 0.01 to 0.5%.

18. A method according to claim 16, wherein the livestock is a poultry and the killed Mycoplasma vaccine is derived from Mycoplasma gallisepecticum. 

1. A SUSPENSION OF KILLED MYCOPLASMA VACCINE FOR IMMUNIZING RESPIRATORY MYCOPLASMOSIS OF LIVESTOCK AND CONSISTING ESSENTIALLY OF FORMALIN KILLED ULTRASONICALLY HOMOGENIZED MYCOPLASMA CELL, A PHYSIOLOGICALLY ACCEPTABLE AQUEOUS MEDIUM OF PH 6.0 TO 7., AND A SUSPENSION STABILIZER.
 2. A suspension of killed Mycoplasma vaccine according to claim 1 wherein said formalin killed and ultrasonically homogenized Mycoplasma vaccine is derived from a formalin-added cell suspension having a formalin concentration of 0.01 to 0.5%.
 3. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the aqueous medium is phosphate buffered saline solution.
 4. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the suspension stabilizer is a saccharide.
 5. A suspension of killed Mycoplasma vaccine according to claim 4, wherein the saccharide is selected from the group consisting of glucose, sUcrose, lactose and dextran.
 6. A suspensison of killed Mycoplasma vaccine according to claim 5, wherein the saccharide is selected from the group consisting of lactose or dextran.
 7. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the suspension stabilizer is an amino acid.
 8. A suspension of killed Mycoplasma vaccine according to claim 7, wherein the amino acid is selected from the group consisting of glutamic acid and glycine.
 9. A suspension of killed Mycoplasma vaccine according to claim 8, wherein the amino acid is glutamic acid.
 10. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the suspension stabilizer is a serum.
 11. A suspension of killed Mycoplasma vaccine according to claim 10, wherein the serum is selected from, the group consisting of chicken serum and chicken serum albumin when the Mycoplasma is Mycoplasma gallisepticum.
 12. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the suspension stabilizer is selected from the group consisting of sorbitan monooleate of polyoxyethylene, gelatin and disodium ethylenediamine tetraacetate.
 13. A suspension of killed Mycoplasma vaccine according to claim 12, wherein the suspension stabilizer is selected from the group consisting of sorbitan monooleate of polyoxyethylene and gelatin.
 14. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the number of the killed Mycoplasma cells is at least 2.0 X 105 per ml of the vaccine.
 15. A suspension of killed Mycoplasma vaccine according to claim 1, wherein the killed Mycoplasma cell is Mycoplasma gallisepticum cell and the livestock is poultry.
 16. A method of immunization for respiratory infections Mycoplasma diseases of livestocks comprising administering a formalin killed and ultrasonically homogenized Mycoplasma vaccine to the livestocks by spraying or injecting the killed vaccine into trachea.
 17. A method according to claim 16 wherein said formalin killed and ultrasonically homogenized Mycoplasma vaccine is derived from a formalin-added cell suspension having a formalin concentration of 0.01 to 0.5%.
 18. A method according to claim 16, wherein the livestock is a poultry and the killed Mycoplasma vaccine is derived from Mycoplasma gallisepecticum. 